The dynamics of secretory vesicles in living hyphae of the pathogen Ustilago maydis.

病原体玉米黑粉菌活菌丝中分泌囊泡的动态。

• 批准号: BB/H019774/1
• 负责人: Gero Steinberg
• 金额: $ 50.27万
• 依托单位: UNIVERSITY OF EXETER
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH019774%2F1
• 关键词: dynamics; secretory; vesicles; living; hyphae

Filamentous fungi are an evolutionarily successful group of organisms of enormous ecological importance as symbionts in mycorrhizal interactions with plants and decomposer of plant debris. They serve in industrial production of proteins and as pathogens pose a threat to public health and agriculture. The basic unit of a filamentous fungus is the hypha; this usually consists of a chain of elongated cells that grow by expansion at the tip, a process called tip growth. This mode of growth allows the invasion of tissue and substrate. Tip growth requires a continuous supply of newly synthesised membranes, proteins and cell wall precursors to the hyphal tip in a process named secretion. The carriers for these supplies are secretory transport vesicles that are taken to the tip where they fuse with the plasma membrane. It is important in the context of this grant application to note that our current view of secretion is that post-Golgi vesicles travel uni-directionally to the hyphal tip where they fuse with the plasma membrane, thereby delivering membranes and proteins. The mechanism by which secretory vesicles are delivered to the hyphal tip is not clear. In analogy to other polarised cells, such as animal neurons, it is assumed that microtubules and kinesin motors mediate long-distance transport of secretory vesicles, whereas short range motility at the plasma membrane is mediated by another system, consisting of myosins and filamentous actin (F-actin). Indeed, recent studies on numerous filamentous fungi, including the plant pathogen Ustilago maydis, demonstrated that microtubules, kinesins, F-actin and myosins are essential for hyphal growth. However, most conclusions from these data are speculative. This due to the fact that motility of secretory cargo was never visualised and no systematic studies on the whole repertoire of motors in a cell have been undertaken. In this project we will address this challenge. We have developed a microscopic setup that allows us to visualise the motility of individual chitin synthase-containing vesicles. We will label numerous other secretory proteins, including secreted enzymes, and will monitor their delivery to the growing hyphal tip. In co-localisation studies using red and green fluorescent proteins we will determine whether cargo travels in the same or different transport vesicles, thereby elucidating the pathways of secretion. We will determine the cytoskeletal elements that underlie vesicle motility and address the role of all kinesin and myosin motors in secretion by making use of existing mutant protein constructs. Finally, we will further investigate the reason for the bi-directional motility of chitosomes. This behaviour is unexpected and we will investigate whether it is a general feature of secretory vesicles. Subsequently, we will use photoactivatable fluorescent proteins to characterise this motility in order to get an insight into the reason for this phenomenon. In summary, we will combine molecular genetics and life cell imaging to: (1) determine the pathways of secretion in the hyphal cell, (2) address the role of 10 kinesins and 4 myosins in motility of secretory vesicles, (3) characterise the bi-directional motility of secretory vesicles in order to get to an understanding the logic behind this behaviour. The expected outcome of this project will be novel insights into the secretory pathway in filamentous fungi. We will provide a comprehensive understanding of the pathways by which cargo reaches the hyphal tip and of the role of kinesins and myosins in delivery of secretory vesicles. If it is discovered that bi-directional motility of secretory vesicles is a general feature, the current paradigm for secretion will have to be modified. This project will therefore be of fundamental interest to all aspects of fungal research, but it will be of particular importance in understanding fungal pathogenicity and industrial production of recombinant proteins.

丝状真菌是一类在进化上取得成功的生物，作为与植物共生的共生体和植物残体的分解者，具有重要的生态意义。它们用于蛋白质的工业生产，并作为病原体对公共卫生和农业构成威胁。丝状真菌的基本单位是菌丝;这通常由一串细长的细胞组成，这些细胞通过尖端的扩张而生长，这一过程称为尖端生长。这种生长模式允许侵入组织和基质。顶端生长需要在一个称为分泌的过程中向菌丝顶端连续供应新合成的膜、蛋白质和细胞壁前体。这些供应的载体是分泌运输囊泡，它们被带到顶端，在那里它们与质膜融合。重要的是，在本授权申请的上下文中注意到，我们目前对分泌的看法是，高尔基体后囊泡单向地行进到菌丝顶端，在那里它们与质膜融合，从而递送膜和蛋白质。分泌囊泡输送到菌丝顶端的机制尚不清楚。与其他极化细胞（如动物神经元）类似，假定微管和驱动蛋白马达介导分泌囊泡的长距离运输，而质膜上的短程运动由另一个系统介导，该系统由肌球蛋白和丝状肌动蛋白（F-肌动蛋白）组成。事实上，最近对许多丝状真菌，包括植物病原体玉米黑粉菌的研究表明，微管，驱动蛋白，F-肌动蛋白和肌球蛋白是菌丝生长所必需的。然而，从这些数据中得出的大多数结论都是推测性的。这是因为分泌性货物的运动性从未被可视化，并且没有对细胞中的整个马达库进行系统的研究。在本项目中，我们将应对这一挑战。我们已经开发了一种显微镜设置，使我们能够可视化的运动的个别几丁质酶含有囊泡。我们将标记许多其他分泌蛋白质，包括分泌酶，并将监测它们的交付日益增长的菌丝尖端。在使用红色和绿色荧光蛋白的共定位研究中，我们将确定货物是否在相同或不同的运输囊泡中旅行，从而阐明分泌途径。我们将利用现有的突变蛋白构建体，确定囊泡运动的细胞骨架要素，并解决所有驱动蛋白和肌球蛋白马达在分泌中的作用。最后，我们将进一步探讨壳聚糖体双向运动的原因。这种行为是出乎意料的，我们将研究它是否是一个分泌囊泡的一般特征。随后，我们将使用光激活荧光蛋白来表征这种运动性，以便深入了解这种现象的原因。总之，我们将结合联合收割机分子遗传学和生命细胞成像：（1）确定菌丝细胞中的分泌途径，（2）解决10个驱动蛋白和4个肌球蛋白在分泌囊泡运动中的作用，（3）研究分泌囊泡的双向运动，以了解这种行为背后的逻辑。该项目的预期成果将是对丝状真菌分泌途径的新见解。我们将提供一个全面的了解的途径，货物到达菌丝顶端和驱动蛋白和肌球蛋白的作用，在交付的分泌囊泡。如果发现分泌囊泡的双向运动是一个普遍特征，那么目前的分泌范式将不得不被修改。因此，该项目将是真菌研究的各个方面的根本利益，但它将是特别重要的了解真菌致病性和重组蛋白的工业生产。

项目成果

Long-distance endosome trafficking drives fungal effector production during plant infection.

• DOI: 10.1038/ncomms6097
• 发表时间: 2014-10-06
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Bielska, Ewa;Higuchi, Yujiro;Schuster, Martin;Steinberg, Natascha;Kilaru, Sreedhar;Talbot, Nicholas J.;Steinberg, Gero
• 通讯作者: Steinberg, Gero

Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase.

• DOI: 10.1038/emboj.2011.361
• 发表时间: 2012-01-04
• 期刊: EMBO JOURNAL
• 影响因子: 11.4
• 作者: Schuster, Martin;Treitschke, Steffi;Kilaru, Sreedhar;Molloy, Justin;Harmer, Nicholas J.;Steinberg, Gero
• 通讯作者: Steinberg, Gero

Kinesin-3 in the basidiomycete Ustilago maydis transports organelles along the entire microtubule array.

担子菌玉米黑粉菌中的驱动蛋白-3 沿着整个微管阵列运输细胞器。

• DOI: 10.1016/j.fgb.2014.10.010
• 发表时间: 2015
• 期刊: FG & B
• 作者: Steinberg G

Early endosome motility spatially organizes polysome distribution.

• DOI: 10.1083/jcb.201307164
• 发表时间: 2014-02-03
• 期刊: The Journal of cell biology
• 作者: Higuchi Y;Ashwin P;Roger Y;Steinberg G
• 通讯作者: Steinberg G